Resistance Genes

ABSTRACT

The present invention relates to polynucleotides encoding adult plant pathogen resistance proteins. Also provided are transgenic plants expressing these polynucleotides to enhance the resistance of the plants to pathogens.

FIELD OF THE INVENTION

The present invention relates to polynucleotides encoding adult plantpathogen resistance proteins. Also provided are transgenic plantsexpressing these polynucleotides to enhance the resistance of the plantsto pathogens.

BACKGROUND OF THE INVENTION

Numerous genes conferring resistance to pathogens have been identifiedand used in plant breeding. However, single-gene pathogen resistance inplants often becomes ineffective due to the emergence of new virulentraces of the disease agent. In contrast, durable disease resistance inplants is generally thought to be controlled by multiple genes.

The wheat (Triticum aestivum) quantitative trait locus, Lr34, providesdurable adult plant resistance to the biotrophic fungi causing thediseases leaf rust, stripe rust, stem rust and powdery mildew (Dyck,1977 and 1987; German and Kolmer, 1992; Bossolini et al. 2006;Spielmeyer et al. 2008). This is despite the limitation that it is noteffective at the seedling stage under normal field conditions. Cultivarswith the resistance locus Lr34 such as Frontana have had effectivedurable resistance to the leaf rust fungus Puccinia triticina Eriks(Dyck et al., 1966; Singh and Rajaram, 1994). To date, isolates of P.triticina with complete virulence to Lr34 have not been detected (Kolmeret al., 2003).

Lr34 resistance has remained genetically inseparable from Yr18 thatconfers resistance to stripe rust (P. striiformis) (Singh, 1992a;McIntosh, 1992). Co-segregation of Lr34/Yr18 with other traits such asleaf tip necrosis (Ltn1), powdery mildew (recently designated Pm38),tolerance to barley yellow dwarf virus (Bdvl) and spot blotch (Bipolarissorokiniana) have been documented (Singh, 1992a, b; McIntosh, 1992;Joshi et al., 2004; Spielmeyer et al., 2005; Liang et al., 2006). Thesemulti-pathogen resistance traits have made the Lr34/Yr18 locus one ofthe most valuable gene regions for disease resistance breeding in wheat.

A few rust resistance genes have been isolated and cloned from wheat(Feuillet et al., 2003; Huang et al., 2003; Cloutier et al., 2007) andother cereals (Collins et al., 1999; Brueggeman et al., 2002) and arepredominantly from the nucleotide binding site-leucine rich repeat(NB-LRR) class of major resistance (R) genes. The only known exceptionis the barley Rpgl rust resistance gene which encodes a protein kinase.These genes encode gene-for-gene resistance against single pathogens andgenerally lead to hypersensitive responses in the plant tissues uponinfection. In contrast, Lr34 confers a broad spectrum resistance againstseveral obligate biotrophic pathogens including fungi from theAscomycetes and Basidiomycetes. Rubiales and Niks (1995) reported thatLr34 is associated with reduced intercellular hyphal growth but not witha hypersensitive response or papilla formation.

The molecular basis of quantitative non-race-specific, adult plantpathogen resistance-type or partial resistance encoded by geneticsystems such as, for example, Lr34 therefore remains unknown.

SUMMARY OF THE INVENTION

The present inventors have identified genes and polypeptides whichconfer enhanced plant pathogen resistance to adult plants.

Accordingly, the present invention provides a transgenic plant which hasintegrated into its genome an exogenous polynucleotide encoding an adultplant pathogen resistance polypeptide and/or an exogenous polynucleotidewhich increases transcription of an endogenous gene encoding an adultplant pathogen resistance polypeptide.

In a preferred embodiment, the plant has accelerated senescence of flagleaf tips when compared to an isogenic plant lacking the exogenouspolynucleotide.

In another preferred embodiment, the plant has enhanced resistance to aplant pathogen when compared to an isogenic plant lacking the exogenouspolynucleotide.

In yet a further preferred embodiment, the polypeptide comprises aminoacids having a sequence as provided in SEQ ID NO:1, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 40%identical, more preferably at least 80% identical, more preferably atleast 90% identical, and even more preferably at least 95% identical, toSEQ ID NO:1. More preferably, the polypeptide comprises amino acidshaving a sequence as provided in SEQ ID NO:1.

In another preferred embodiment, the polynucleotide comprisesnucleotides having a sequence as provided in SEQ ID NO:2, a sequencewhich is at least 40% identical to SEQ ID NO:2, and/or a sequence whichhybridizes to SEQ ID NO:2.

In another embodiment, the exogenous polynucleotide which increasestranscription of an endogenous gene encoding an adult plant pathogenresistance polypeptide is a genetic element, such as a promoter, whichenhances the function of the endogenous gene promoter. Alternatively,the exogenous polynucleotide which increases transcription of anendogenous gene encoding an adult plant pathogen resistance polypeptideencodes a transcription factor which enhances expression of theendogenous gene.

Preferably, the plant is a cereal plant. Examples of transgenic cerealplants of the invention include, but are not limited to wheat, barley,maize, rice, oats and triticale. In a particularly preferred embodiment,the plant is wheat.

Examples of plant pathogens include, but are not limited to viruses,bacteria and fungi.

In a preferred embodiment, the pathogen is a biotrophic fungus. Examplesof biotrophic fungi include, but are not limited to, Fusariumgraminearum (which causes head blight), Erysiphe graminis f. sp. tritici(which causes powdery mildew), Bipolaris sorokiniana (which causes spotblotch), Puccinia graminis f. sp. tritici (which causes stem rust),Puccinia striiformis (which causes stripe rust) and Puccinia reconditef. sp. tritici (which causes leaf rust).

In an embodiment, the pathogen is barley yellow dwarf virus (BYDV).

In an embodiment, the plant comprises one or more further exogenouspolynucleotides encoding a plant pathogen resistance polypeptide.Examples of such genes include, but are not limited to, Lr1, Lr3, Lr2a,Lr3ka, Lr1l, Lr13, Lr16, Lr17, Lr18, Lr21 and LrB.

In another aspect, the present invention provides a process foridentifying a polynucleotide encoding a plant pathogen resistancepolypeptide comprising:

(i) obtaining a polynucleotide operably linked to a promoter, thepolynucleotide encoding a polypeptide comprising amino acids having asequence as provided in SEQ ID NO:1, a biologically active fragmentthereof, or an amino acid sequence which is at least 40% identical toSEQ ID NO:1,

(ii) introducing the polynucleotide into a plant,

(iii) determining whether the level of resistance to a plant pathogen ismodified relative to an isogenic plant lacking the polynucleotide, and

(iv) optionally, selecting a polynucleotide which when expressedenhances resistance to the plant pathogen.

Preferably, the polynucleotide comprises nucleotides having a sequenceas provided in SEQ ID NO:2, a sequence which is at least 40% identicalto SEQ ID NO:2, and/or a sequence which hybridizes to SEQ ID NO:2.

Preferably, the plant is a cereal plant.

Preferably, the cereal plant is a wheat plant.

In a preferred embodiment, the polypeptide is a plant polypeptide ormutant thereof.

In a further embodiment, step (ii) further comprises stably integratingthe polynucleotide operably linked to a promoter into the genome of theplant.

In yet another aspect, the present invention provides a substantiallypurified and/or recombinant adult plant pathogen resistance polypeptide.

In a preferred embodiment, the polypeptides comprises amino acids havinga sequence as provided in SEQ ID NO:1, a biologically active fragmentthereof, or an amino acid sequence which is at least 40% identical, morepreferably at least 80% identical, more preferably at least 90%identical, and even more preferably at least 95% identical, to SEQ IDNO:1.

In a preferred embodiment, the polypeptide lacks a phenylalanine residueor any amino acid at a position corresponding to amino acid number 546of SEQ ID NO:4.

In another preferred embodiment, the polypeptide has an amino acid otherthan a tyrosine residue at a position corresponding to amino acid number634 of SEQ ID NO:4. More preferably, the polypeptide comprises ahistidine residue at a position corresponding to amino acid number 634of SEQ ID NO:4.

Also provided is a fusion protein further comprising at least one otherpolypeptide sequence. The at least one other polypeptide may be, forexample, a polypeptide that enhances the stability of a polypeptide ofthe present invention, or a polypeptide that assists in the purificationor detection of the fusion protein.

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide comprising nucleotides having a sequence asprovided in SEQ ID NO:2, a sequence which is at least 40% identical toSEQ ID NO:2, a sequence encoding a polypeptide of the invention, and/ora sequence which hybridizes to SEQ ID NO:2.

Preferably, the polynucleotide comprises a sequence of nucleotides whichhybridizes to SEQ ID NO:2 under stringent conditions.

Preferably, the polynucleotide hybridizes along the full length of apolynucleotide consisting of nucleotides having the sequence of SEQ IDNO:2.

Preferably, the polynucleotide encodes an adult plant pathogenresistance polypeptide.

In a further aspect, the present invention provides a chimeric vectorcomprising the polynucleotide of the invention.

Preferably, the polynucleotide is operably linked to a promoter.

In a further aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide of the invention and/or a vectorof the invention.

The cell can be any cell type such as, but not limited to, a plant cell,a bacterial cell, an animal cell or a yeast cell.

Preferably, the cell is a plant cell. More preferably, the plant cell isa cereal plant cell. Even more preferably, the cereal plant cell is awheat cell.

In a further aspect, the present invention provides a method ofproducing the polypeptide of the invention, the method comprisingexpressing in a cell or cell free expression system the polynucleotideof the invention.

Preferably, the method further comprises isolating the polypeptide.

In yet another aspect, the present invention provides a transgenicnon-human organism comprising an exogenous polynucleotide of theinvention, a vector of the invention and/or a recombinant cell of theinvention.

Preferably, the transgenic non-human organism is a plant.

In another aspect, the present invention provides a method of producingthe cell of the invention, the method comprising the step of introducingthe polynucleotide of the invention, or a vector of the invention, intoa cell.

Preferably, the cell is a plant cell.

In a further aspect, the present invention provides a method ofproducing a transgenic plant, the method comprising regenerating atransgenic plant from the cell of the invention.

Also provided is the use of the polynucleotide of the invention, or avector of the invention, to produce a recombinant cell.

Further, provided is the use of the polynucleotide of the invention, ora vector of the invention, to produce a transgenic plant.

Preferably, the transgenic plant has accelerated senescence of flag leaftips when compared to an isogenic plant lacking the exogenouspolynucleotide and/or vector, and/or has enhanced resistance to a plantpathogen when compared to an isogenic plant lacking the exogenouspolynucleotide and/or vector.

In another aspect, the present invention provides a transgenic plant, orprogeny thereof, produced using a method of the invention.

In a further aspect, the present invention provides a plant part of theplant of the invention.

Examples of such plant parts include, but are not limited to, leaves,roots, stems and/or seeds. In a preferred embodiment, the plant part isa seed that comprises an exogenous polynucleotide encoding an adultplant pathogen resistance polypeptide.

In another aspect, the present invention provides a method of producinga plant part, the method comprising,

a) growing a plant of the invention, and

b) harvesting the plant part.

In yet a further aspect, the present invention provides a method ofproducing flour, wholemeal, starch or other product obtained from seed,the method comprising;

a) obtaining seed of the invention, and

b) extracting the flour, wholemeal, starch or other product.

In another aspect, the present invention provides a product producedfrom a plant of the invention and/or a plant part of the invention.

In one embodiment, the product is a food product. Examples include, butare not limited to, flour, starch, leavened or unleavened breads, pasta,noodles, animal fodder, breakfast cereals, snack foods, cakes, malt,beer, pastries and foods containing flour-based sauces.

In another embodiment, the product is a non-food product. Examplesinclude, but are not limited to, films, coatings, adhesives, buildingmaterials and packaging materials.

In a further aspect, the present invention provides a method ofpreparing a food product of the invention, the method comprising mixingseed, or flour, wholemeal or starch from said seed, with anotheringredient.

In a further aspect, the present invention provides a method ofpreparing malt, comprising the step of germinating the seed of theinvention.

In another embodiment, the present invention provides a compositioncomprising a polypeptide of the invention, a polynucleotide of theinvention, a vector of the invention, and/or recombinant cell of theinvention, and one or more acceptable carriers.

In another aspect, the present invention provides a substantiallypurified antibody, or fragment thereof, that specifically binds apolypeptide of the invention.

Also provided is a method of identifying a compound that binds to apolypeptide comprising amino acids having a sequence as provided in SEQID NO:1 or SEQ ID NO:4, a biologically active fragment thereof, or anamino acid sequence which is at least 40% identical to SEQ ID NO:1and/or SEQ ID NO:4, the method comprising:

i) contacting the polypeptide with a candidate compound, and

ii) determining whether the compound binds the polypeptide.

Further, provided is a method of identifying a compound that istransported across a cell membrane by a polypeptide comprising aminoacids having a sequence as provided in SEQ ID NO:1 or SEQ ID NO:4, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to SEQ ID NO:1 and/or SEQ ID NO:4, the methodcomprising:

i) contacting the polypeptide present in a cell membrane with acandidate compound,

ii) determining whether the polypeptide transports the compound acrossthe cell membrane.

Preferably, the polypeptide is expressed in a cell.

Preferably, the cell is a plant cell.

In an embodiment, the method further comprises comparing the binding,and/or transport, of the compound to a first polypeptide comprising anamino acid sequence provided as SEQ ID NO:1 to a second polypeptidecomprising an amino acid sequence provided as SEQ ID NO:4.

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide which, when present in a cell of a plant,decreases the expression of at least one gene that hybridises understringent conditions to a nucleic acid molecule encoding a polypeptidecomprising amino acids having a sequence as provided in SEQ ID NO:1 orSEQ ID NO:4, a biologically active fragment thereof, or an amino acidsequence which is at least 40% identical to SEQ ID NO:1 and/or SEQ IDNO:4, said decreased expression being relative to an otherwise isogeniccell of a plant that lacks said polynucleotide.

In an embodiment, the polynucleotide encodes an adult plant pathogenresistance polypeptide.

Preferably, the polynucleotide of this aspect is operably linked to apromoter capable of directing expression of the polynucleotide in a cellof a plant.

Preferably, the polynucleotide of this aspect is an antisensepolynucleotide, a sense polynucleotide, a catalytic polynucleotide, anartificial microRNA or a duplex RNA molecule.

In a further aspect, the present invention provides a method ofidentifying a plant comprising a gene encoding an adult plant pathogenresistance polypeptide, the method comprising

i) amplifying and/or sequencing, from a sample of the plant, at least aportion of a polynucleotide which encodes a polypeptide comprising aminoacids having a sequence as provided in SEQ ID NO:1 or SEQ ID NO:4, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to SEQ ID NO:1 and/or SEQ ID NO:4,

ii) determining if the plant comprises a polynucleotide encoding anadult plant pathogen resistance polypeptide.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Consensus genetic map of wheat chromosome 7D including Lr34based on three high-resolution mapping populations defined a 0.15 cMtarget interval for Lr34 between XSWSNP3 and XcsLVE17. Relativepositions of molecule markers are shown together with the observedrecombinational distances in cM.

FIG. 2. Schematic of expanded view of part of wheat chromosome 7DSbetween XSWSNP3 and XcsLVE17 showing relative positions of open readingframes. The corresponding physical target interval sequenced on the+Lr34 cultivar ‘Chinese Spring’ contained ten candidate genes, nine ofwhich are represented in the Figure by arrows. Numbers refer to therespective nucleotide positions within the 420 kb sequenced interval.Abbreviations: Gly, glycosyl transferase; Cyst, cysteine proteinase;Cyt, Cytochrome P450; LecK, lectin kinase; ABC, ABC transporter; Hex,hexose carrier.

FIG. 3. Gene structure of Lr34. Open boxes indicate exons, while intronsare shown as adjoining lines. Marks indicate the positions of mutationsites of the mutants labelled 2B, 2F, 2G, 3E, 4C, 4D, m19 and m21. Thethree sequence differences between susceptible and resistant alleles ofLr34 are indicated: +Lr34 resistant allele from Chinese Spring, −Lr34susceptible allele from Renan.

FIG. 4. Lr34 protein sequence and polymorphisms between resistant andsusceptible cultivars. Amino acid sequence of the Lr34 protein(susceptible allele) from cultivar ‘Renan’. The two amino acids that arealtered in the resistant allele are highlighted. Other boxes indicatethe positions of the highly conserved motifs within the nucleotidebinding domains. Motifs: “Walker A” GPPGCGKS (amino acids 168-175) (SEQID NO:50) and GVSGAGKT (amino acids 847-854) (SEQ ID NO:51); “ABCsignature” ISGGQKKRLTTA (amino acids 307-318) (SEQ ID NO:52) andLSMEQRKRLTIA (amino acids 954-965) (SEQ ID NO:53); “Walker B” AYFMD(amino acids 327-331) (SEQ ID NO:54) and IILMD (amino acids 974-978)(SEQ ID NO:55). Amino acid changes in the resistant allele of Lr34 inwheat cultivar Chinese Spring are deletion of amino acid 546 (Phe (F))and substitution of amino acid 634 (tyrosine (Y)) to histidine.Underlined portions are the two transmembrane domains (amino acids502-750 and 1152-1392).

FIG. 5. Schematic representation of the Lr34 protein showing the twonucleotide binding domains (NBD) and the two transmembrane domains. Thetwo diagnostic polymorphisms between resistant and susceptible allelesin the first transmembrane domain are indicated by stars.

FIG. 6. Lr34 amino acid sequence alignment. Alignment of Lr34 ofcultivar Renan with rice PDR23 (Os12g0512700) (SEQ ID NO:47) andArabidopsis PDR5 (At3g53480) (SEQ ID NO:48) and PDR9 (At2g37280) (SEQ IDNO:49). Residues identical in all the four transporters are indicated.Rice PDR23 has been newly annotated according to the wheat Lr34 cDNA.

FIG. 7. Expression analysis of Lr34. Semi-quantitative RT-PCR using aprobe from the 5′ end of the gene. Leaves of the near isogenic lines‘Thatcher’ and ‘Thatcher Lr34’ were harvested at the seedling stageafter 14 days and of adult flag leaves on 53 and 63 days old plants.Adult leaves were halved to separately study expression levels of leafbase and leaf tip. Abbreviations: TH=‘Thatcher’; TH Lr34=‘ThatcherLr34’; GAPDH=Glyceraldehyde 3-phosphate dehydrogenase.

FIG. 8. Lr34 regulates senescence of flag leaves. Northern Blot usingHvS40 on 63 days old flag leaves of the near isogenic lines ‘Thatcher’and ‘Thatcher Lr34’ and the azide induced Lr34 mutants 2B, 2F, 2G, 3E,4C and 4E. TH=‘Thatcher’; TH Lr34=‘ Thatcher Lr34’.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Amino acid sequence of Lr34 protein (resistant allele) fromTriticum aestivum cv Chinese spring.SEQ ID NO:2—Nucleotide coding sequence for Lr34 from Triticum aestivumcv Chinese spring.SEQ ID NO:3—Nucleotide sequence of the Lr34 gene (genomic sequence) fromTriticum aestivum cv Chinese spring. 24 exons are present which encodethe Lr34 protein:exon 1 starts at nucleotide 3042 and ends at nucleotide 3316;exon 2 starts at nucleotide 3416 and ends at nucleotide 3539;exon 3 starts at nucleotide 3693 and ends at nucleotide 3778;exon 4 starts at nucleotide 3934 and ends at nucleotide 4018;exon 5 starts at nucleotide 6527 and ends at nucleotide 6686;exon 6 starts at nucleotide 6784 and ends at nucleotide 6860;exon 7 starts at nucleotide 7119 and ends at nucleotide 7172;exon 8 starts at nucleotide 7271 and ends at nucleotide 7361;exon 9 starts at nucleotide 7439 and ends at nucleotide 7740;exon 10 starts at nucleotide 7833 and ends at nucleotide 8108;exon 11 starts at nucleotide 8187 and ends at nucleotide 8497;exon 12 starts at nucleotide 8583 and ends at nucleotide 8743;exon 13 starts at nucleotide 8825 and ends at nucleotide 8928;exon 14 starts at nucleotide 9015 and ends at nucleotide 9168;exon 15 starts at nucleotide 9606 and ends at nucleotide 9513;exon 16 starts at nucleotide 9808 and ends at nucleotide 9581;exon 17 starts at nucleotide 9985 and ends at nucleotide 10317;exon 18 starts at nucleotide 10427 and ends at nucleotide 10717;exon 19 starts at nucleotide 12159 and ends at nucleotide 12242;exon 20 starts at nucleotide 12711 and ends at nucleotide 12844;exon 21 starts at nucleotide 12995 and ends at nucleotide 13222;exon 22 starts at nucleotide 13318 and ends at nucleotide 13489;exon 23 starts at nucleotide 13569 and ends at nucleotide 13823; andexon 24 starts at nucleotide 14613 and ends at nucleotide 14939.SEQ ID NO:4—Amino acid sequence of Lr34 protein (susceptible allele)from Triticum aestivum “Renan”.SEQ ID NO:5—Nucleotide coding sequence for Lr34 (susceptible allele)from Triticum aestivum “Renan”.SEQ ID NO:6—Genomic DNA for Aegilops tauschii Lr34 equivalent. Codingregion starts at nucleotide 2426 and ends at nucleotide 14212.SEQ ID NO:7—EST of Triticum aestivum Lr34 (GenBank Accession No.CJ669561).SEQ ID NO:8—EST of Triticum aestivum Lr34 (GenBank Accession No.DR733734).SEQ ID NO:9—EST of Triticum aestivum Lr34 (GenBank Accession No.CJ562397).SEQ ID NO:10—EST of Triticum aestivum Lr34 (GenBank Accession No.CV773074).SEQ ID NO:11—EST for Hordeum vulgare Lr34 (GenBank Accession No.BU991506).SEQ ID NO's: 12-46—Oligonucleotide primers.SEQ ID NO:47—Rice ABC transporter PDR23.SEQ ID NO:48—Arabidopsis thaliana ABC transporter PDR5.SEQ ID NO:49—Arabidopsis thaliana ABC transporter PDR9.SEQ ID NO: 50—N-terminal Walker A sequence of Lr34.SEQ ID NO: 51—C-terminal Walker A sequence of Lr34.SEQ ID NO: 52—N-terminal ABC signature sequence of Lr34.SEQ ID NO: 53—C-terminal ABC signature sequence of Lr34.SEQ ID NO: 54—N-terminal Walker B sequence of Lr34.SEQ ID NO: 55—C-terminal Walker B sequence of Lr34.SEQ ID NO: 56—Consensus Walker A sequence of ABC transporters.SEQ ID NO: 57—Consensus Walker B sequence of ABC transporters.SEQ ID NO: 58—Consensus ABC signature sequence of ABC transporters.SEQ ID NO:59—PDR signature sequence 1.SEQ ID NO:60—PDR signature sequence 2.SEQ ID NO:61—PDR signature sequence 3.SEQ ID NO:62—PDR signature sequence 4.SEQ ID NO:63—Polypeptide encoded by Lr34 homeolog on wheat chromosome7B.SEQ ID NO:64—Open reading frame encoding Lr34 homeolog on wheatchromosome 7B.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, plant molecular biology, protein chemistry, andbiochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

Polypeptides/Peptides

By “substantially purified polypeptide” or “purified polypeptide” wemean a polypeptide that has generally been separated from the lipids,nucleic acids, other peptides, and other contaminating molecules withwhich it is associated in its native state. Preferably, thesubstantially purified polypeptide is at least 90% free from othercomponents with which it is naturally associated.

The term “recombinant” in the context of a polypeptide refers to thepolypeptide when produced by a cell, or in a cell-free expressionsystem, in an altered amount or at an altered rate compared to itsnative state. In one embodiment, the cell is a cell that does notnaturally produce the polypeptide. However, the cell may be a cell whichcomprises a non-endogenous gene that causes an altered amount of thepolypeptide to be produced. A recombinant polypeptide of the inventionincludes polypeptides which have not been separated from othercomponents of the transgenic (recombinant) cell, or cell-free expressionsystem, in which it is produced, and polypeptides produced in such cellsor cell-free systems which are subsequently purified away from at leastsome other components. In an embodiment, a “recombinant polypeptide” isa polypeptide made by the expression of a recombinant polynucleotide ina cell, preferably a plant cell and more preferably a cereal plant cell.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

As used herein, the term “adult plant pathogen resistance polypeptide”refers to a protein encoded by a gene which ordinarily confers upon anadult plant an enhanced resistance to a plant pathogen when compared toan isogenic plant lacking said gene, and which confers on seedlings ofthe same plant substantially less or no resistance to the same pathogenwhen the plant is grown in normal field conditions. This term alsorefers to the naturally produced protein (or wild type protein fromwhich a mutant protein is derived) encoded by a gene conferring upon anadult plant (for example, of the wheat cultivar Frontana), but not aseedling, when grown in normal field conditions, enhanced resistance toa plant pathogen. Typically, adult plant pathogen resistancepolypeptides do not confer a hypersensitive response on the plants inthe presence of the pathogen, and the resistance is durable in the fieldover time. As used herein, “adult plant” refers to a plant that hascommenced the reproductive phase of growth and development. In anembodiment, less than half of the protein is produced per gram dryweight in leaves of a seedling when compared to leaves of the adultplant. Examples of plant pathogens for which resistance is enhancedinclude, but are not limited to, Fusarium graminearum, Erysiphe graminisf. sp. tritici, Bipolaris sorokiniana, Puccinia graminis f. sp. tritici,Puccinia striiformis and Puccinia recondite f. sp. tritici.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 150 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 150 amino acids. More preferably, the query sequenceis at least 500 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 500 amino acids. Morepreferably, the query sequence is at least 1,000 amino acids in lengthand the GAP analysis aligns the two sequences over a region of at least1,000 amino acids. Even more preferably, the query sequence is at least1,250 amino acids in length and the GAP analysis aligns the twosequences over a region of at least 1,250 amino acids. Even morepreferably, the GAP analysis aligns two sequences over their entirelength.

As used herein a “biologically active” fragment is a portion of apolypeptide of the invention which maintains a defined activity of thefull-length polypeptide. Biologically active fragments can be any sizeas long as they maintain the defined activity but are preferably atleast 1000 or at least 1200 amino acid residues long. Preferably, thebiologically active fragment maintains at least 10% of the activity ofthe full length protein.

The phrase “enhanced resistance to a plant pathogen” is used herein as arelative term such that a plant of the invention has an increased levelof resistance to a plant pathogen when compared to a geneticallyidentical lacking the exogenous polynucleotide. Enhanced resistance canbe determined by a number of methods known in the art such as analysingthe plants for the amount of pathogen and/or analysing plant growth orthe amount of damage to a plant in the presence of the pathogen.

As used herein, the term “has accelerated senescence of flag leaf tips”refers to an early onset of aging of the extremity of the lowermost leafon the stem of a plant. This is used herein as a relative term such thata plant of the invention has an increased senescence of flag leaf tipswhen compared to a genetically identical flag leaf lacking the exogenouspolynucleotide. Accelerated senescence of flag leaf tips can be measuredby any means known in the art, such as that described in Example 5.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

As used herein, the phrase “at a position corresponding to amino acidnumber” or variations thereof refers to the relative position of theamino acid compared to surrounding amino acids. In this regard, in someembodiments a polypeptide of the invention may have deletional orsubstitutional mutations which alters the relative positioning of theamino acid when aligned against, for instance, SEQ ID NO:1 and/or SEQ IDNO:4. For example, the polypeptide with a sequence as provided in SEQ IDNO:1 has a single amino acid deletion when compared to the polypeptidewith a sequence as provided in SEQ ID NO:4, namely the phenylalanine atposition number 546 of SEQ ID NO:4 is missing in SEQ ID NO:1 and has notbe substituted with another amino acid. As a result, the skilled personwill appreciate that amino acid number 634 of SEQ ID NO:4 (Y)corresponds to amino acid number 633 of SEQ ID NO:4 (H).

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics. Preferred amino acid sequencemutants have only one, two, three, four or less than 10 amino acidchanges relative to the reference wildtype polypeptide.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide of the invention can be subjectedto in vitro mutagenesis. Such in vitro mutagenesis techniques includesub-cloning the polynucleotide into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. In another example, the polynucleotides of the inventionare subjected to DNA shuffling techniques as broadly described byHarayama (1998). Products derived from mutated/altered DNA can readilybe screened using techniques described herein to determine if theypossess pathogen resistance and/or ABC transporter activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various strains orspecies are identical. These positions may be important for biologicalactivity. These sites, especially those falling within a sequence of atleast three other identically conserved sites, are preferablysubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 1 under the heading of “exemplarysubstitutions”.

In a preferred embodiment a mutant/variant polypeptide has one or two orthree or four conservative amino acid changes when compared to anaturally occurring polypeptide. Details of conservative amino acidchanges are provided in Table 1. In a preferred embodiment, the changesare not in one or more of the motifs which are highly conserved betweenthe different polypeptides provided herewith. As the skilled personwould be aware, such minor changes can reasonably be predicted not toalter the activity of the polypeptide when expressed in a recombinantcell.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In an embodiment, the protein of the invention is a PDR (pleiotropicdrug resistance homolog) ABC transporter and comprises two nucleotidebinding domains (NBD) and two transmembrane domains configured as shownin FIG. 5.

The primary amino acid sequence of Lr34 can be used to designvariants/mutants thereof based on comparisons with closely related ABCtransporters. As the skilled addressee will appreciate, residues highlyconserved amongst closely related PDR ABC transporters are less likelyto be able to be altered, especially with non-conservativesubstitutions, and activity maintained than less conserved residues.Such conserved regions and possible substitutions are described by Rae(2007), van den Brule and Smart (2002) and Verrier et al. (2008). Thepolypeptide generally comprises two Walker A boxes (GX₄GK[ST]) (SEQ IDNO:56) (corresponds to SEQ ID NO's: 50 and 51 of Lr34) and two Walker Bboxes ((hydrophobic)₄[DE]) (SEQ ID NO:57) (corresponds to SEQ ID NO's:54 and 55 of Lr34), and two ABC signature motifs([LIVMFY]S[SGM][GE]X₃[RKA][LIVMYA]X[LIVFMT][AG]) (SEQ ID NO:58)(corresponds to SEQ ID NO's: 52 and 53 of Lr34), with each NBDcomprising, in order from the N-terminus, a Walker A, ABC signature andWalker B motif (see, for example, FIG. 4). In the above sequences X maybe any amino acid, and may be independently the same or different.

Furthermore, the polypeptide generally comprises a PDR signature 1(LLLGPP) (SEQ ID NO:59) which is immediately N-terminal to and slightlyoverlaps with the N-terminal Walker A box; PDR signature 2 (GLDSST) (SEQID NO:60) which starts about four residues C-terminal to the N-terminalWalker B box; PDR signature 3 (GLD[AT]R[AS]AAIV[MI]R) (SEQ ID NO:61)which starts about four residues C-terminal to the C-terminal Walker Bbox; and PDR signature 4 (VCTIHQPS) (SEQ ID NO:62) which starts about 86residues C-terminal to PDR signature 3.

In an embodiment, the polypeptide of the invention comprises one or moreof the amino acids motifs provided as SEQ ID NO's: 56 to 58, preferablytwo copies of all three. More preferably, the polypeptide of theinvention comprises one or more of the amino acids motifs provided asSEQ ID NO's: 50 to 55, preferably all six.

In addition, in yet a further embodiment the polypeptide of theinvention comprises one or more of the amino acids motifs provided asSEQ ID NO's: 59 to 62, preferably all four.

Sources of naturally occurring variants of SEQ ID NO:1 which conferresistance as described herein are outlined in Table 5. Based on theinformation provided herein, the skilled person could readily determinethe amino acid sequence of these naturally occurring variants, as wellas polynucleotides encoding therefor.

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. The polypeptides may bepost-translationally modified in a cell, for example by phosphorylation,which may modulate its activity. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural polypeptides,production and recovery of recombinant polypeptides, and chemicalsynthesis of the polypeptides. In one embodiment, an isolatedpolypeptide of the present invention is produced by culturing a cellcapable of expressing the polypeptide under conditions effective toproduce the polypeptide, and recovering the polypeptide. A preferredcell to culture is a recombinant cell of the present invention.Effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitpolypeptide production. An effective medium refers to any medium inwhich a cell is cultured to produce a polypeptide of the presentinvention. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art. Apreferred means of producing the polypeptides is in a transgenic plant,preferably a transgenic cereal plant.

Polynucleotides and Genes

The present invention refers to various polynucleotides. As used herein,a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA or a combinationthereof, and includes mRNA, cRNA, cDNA, tRNA, siRNA, shRNA and hpRNA. Itmay be DNA or RNA of cellular, genomic or synthetic origin, for examplemade on an automated synthesizer, and may be combined with carbohydrate,lipids, protein or other materials, labelled with fluorescent or othergroups, or attached to a solid support to perform a particular activitydefined herein, or comprise one or more modified nucleotides not foundin nature, well known to those skilled in the art. The polymer may besingle-stranded, essentially double-stranded or partly double-stranded.An example of a partly-double stranded RNA molecule is a hairpin RNA(hpRNA), short hairpin RNA (shRNA) or self-complementary RNA whichinclude a double stranded stem formed by basepairing between anucleotide sequence and its complement and a loop sequence whichcovalently joins the nucleotide sequence and its complement. Basepairingas used herein refers to standard basepairing between nucleotides,including G: U basepairs. “Complementary” means two polynucleotides arecapable of basepairing (hybridizing) along part of their lengths, oralong the full length of one or both. A “hybridized polynucleotide”means the polynucleotide is actually basepaired to its complement. Theterm “polynucleotide” is used interchangeably herein with the term“nucleic acid”.

By “isolated polynucleotide” we mean a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state. Preferably, the isolatedpolynucleotide is at least 90% free from other components with which itis naturally associated.

The present invention involves modification of gene activity and theconstruction and use of chimeric genes. As used herein, the term “gene”includes any deoxyribonucleotide sequence which includes a proteincoding region or which is transcribed in a cell but not translated, aswell as associated non-coding and regulatory regions. Such associatedregions are typically located adjacent to the coding region or thetranscribed region on both the 5′ and 3′ ends for a distance of about 2kb on either side. In this regard, the gene may include control signalssuch as promoters, enhancers, termination and/or polyadenylation signalsthat are naturally associated with a given gene, or heterologous controlsignals in which case the gene is referred to as a “chimeric gene”. Thesequences which are located 5′ of the coding region and which arepresent on the mRNA are referred to as 5′ non-translated sequences. Thesequences which are located 3′ or downstream of the coding region andwhich are present on the mRNA are referred to as 3′ non-translatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene.

A “Lr34 gene” as used herein refers to a nucleotide sequence which ishomologous to the isolated Lr34 gene (SEQ ID NO:3) or Lr34 cDNA (SEQ IDNO:2) described herein, which encodes a protein that confers resistanceto a pathogen, preferably a fungal pathogen, on a plant, preferably acereal plant and more preferably a wheat plant. Preferably, the proteinconfers resistance to more than one fungal pathogen. Lr34 genes includethe naturally occurring alleles or variants existing in cereals such aswheat, including those encoded by the D genomes of hexaploid wheat andits D genome diploid progenitors or relatives, as well as non-naturallyoccurring variants which may be produced by those skilled in the art ofgene modification. Nucleic acid molecules having the nucleotide sequenceshown herein as SEQ ID NO:2 (cDNA) or SEQ ID NO:3 (genomic sequence),encoding a protein with amino acid sequence SEQ ID NO:1, are examples ofan Lr34 gene. In a preferred embodiment, a Lr34 gene refers to a nucleicacid molecule comprising nucleotides having a sequence having at least90% identity to SEQ ID NO:2.

A genomic form or clone of a gene containing the transcribed region maybe interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” An “intron” as usedherein is a segment of a gene which is transcribed as part of a primaryRNA transcript but is not present in the mature mRNA molecule. Intronsare removed or “spliced out” from the nuclear or primary transcript;introns therefore are absent in the messenger RNA (mRNA). Introns maycontain regulatory elements such as enhancers. “Exons” as used hereinrefer to the DNA regions corresponding to the RNA sequences which arepresent in the mature mRNA or the mature RNA molecule in cases where theRNA molecule is not translated. An mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.The term “gene” includes a synthetic or fusion molecule encoding all orpart of the proteins of the invention described herein and acomplementary nucleotide sequence to any one of the above. A gene may beintroduced into an appropriate vector for extrachromosomal maintenancein a cell or for integration into the host genome.

As used herein, a “chimeric gene” refers to any gene that is not anative gene in its native location. Typically, a chimeric gene comprisesregulatory and transcribed or protein coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. The term “endogenous” is used herein to refer to a substancethat is normally present or produced in an unmodified plant at the samedevelopmental stage as the plant under investigation. An “endogenousgene” refers to a native gene in its natural location in the genome ofan organism. As used herein, “recombinant nucleic acid molecule”,“recombinant polynucleotide” or variations thereof refer to a nucleicacid molecule which has been constructed or modified by recombinant DNAtechnology. The terms “foreign polynucleotide” or “exogenouspolynucleotide” or “heterologous polynucleotide” and the like refer toany nucleic acid which is introduced into the genome of a cell byexperimental manipulations. For example, the present inventors haveidentified the Lr34 homeolog on wheat chromosome 7B (see SEQ ID NO's 63and 64). The skilled person can use this information to mutant the Lr34gene homeolog in durum wheat such that it encodes a protein of theinvention which lacks a phenylalanine residue or any amino acid at aposition corresponding to amino acid number 546 of SEQ ID NO:4, and hasan amino acid other than a tyrosine residue at a position correspondingto amino acid number 634 of SEQ ID NO:4. Such a mutated gene, and theencoded mRNA, would be considered as an “exogenous” polynucleotide ofthe invention.

Foreign or exogenous genes may be genes that are inserted into anon-native organism, native genes introduced into a new location withinthe native host, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The term“genetically modified” includes introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny.

Furthermore, the term “exogenous” in the context of a polynucleotide(nucleic acid) refers to the polynucleotide when present in a cell, orin a cell-free expression system, in an altered amount compared to itsnative state. In one embodiment, the cell is a cell that does notnaturally comprise the polynucleotide. However, the cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 450nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 450 nucleotides. Preferably, the querysequence is at least 1,500 nucleotides in length, and the GAP analysisaligns the two sequences over a region of at least 1,500 nucleotides.Even more preferably, the query sequence is at least 3,000 nucleotidesin length and the GAP analysis aligns the two sequences over a region ofat least 3,000 nucleotides. Even more preferably, the GAP analysisaligns two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In a preferred embodiment, the polynucleotide of the invention is not asequence of nucleotides as provided in any one of SEQ ID NO's 7 to 11.

In a further embodiment, the present invention relates topolynucleotides which are substantially identical to those specificallydescribed herein. As used herein, with reference to a polynucleotide theterm “substantially identical” means the substitution of one or a few(for example 2, 3, or 4) nucleotides whilst maintaining at least oneactivity of the native protein encoded by the polynucleotide. Inaddition, this term includes the addition or deletion of nucleotideswhich results in the increase or decrease in size of the encoded nativeprotein by one or a few (for example 2, 3, or 4) amino acids whilstmaintaining at least one activity of the native protein encoded by thepolynucleotide.

The present invention refers to use of oligonucleotides. As used herein,“oligonucleotides” are polynucleotides up to 50 nucleotides in length.The minimum size of such oligonucleotides is the size required for theformation of a stable hybrid between an oligonucleotide and acomplementary sequence on a nucleic acid molecule of the presentinvention. They can be RNA, DNA, or combinations or derivatives ofeither. Oligonucleotides are typically relatively short single strandedmolecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length.When used as a probe or as a primer in an amplification reaction, theminimum size of such an oligonucleotide is the size required for theformation of a stable hybrid between the oligonucleotide and acomplementary sequence on a target nucleic acid molecule. Preferably,the oligonucleotides are at least 15 nucleotides, more preferably atleast 18 nucleotides, more preferably at least 19 nucleotides, morepreferably at least 20 nucleotides, even more preferably at least 25nucleotides in length. Oligonucleotides of the present invention used asa probe are typically conjugated with a label such as a radioisotope, anenzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

The present invention includes oligonucleotides that can be used as, forexample, probes to identify nucleic acid molecules, or primers toproduce nucleic acid molecules. Probes and/or primers can be used toclone homologues of the polynucleotides of the invention from otherspecies. Furthermore, hybridization techniques known in the art can alsobe used to screen genomic or cDNA libraries for such homologues.

Polynucleotides and oligonucleotides of the present invention includethose which hybridize under stringent conditions to a sequence providedas SEQ ID NO's: 2 and/or 3. As used herein, stringent conditions arethose that (1) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄at 50° C.; (2) employ during hybridisation a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextransulfate at 42° C. in 0.2×SSC and 0.1% SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid). A variant of a polynucleotide or anoligonucleotide of the invention includes molecules of varying sizes of,and/or are capable of hybridising to, the wheat genome close to that ofthe reference polynucleotide or oligonucleotide molecules definedherein. For example, variants may comprise additional nucleotides (suchas 1, 2, 3, 4, or more), or less nucleotides as long as they stillhybridise to the target region. Furthermore, a few nucleotides may besubstituted without influencing the ability of the oligonucleotide tohybridise to the target region. In addition, variants may readily bedesigned which hybridise close to, for example to within 50 nucleotides,the region of the plant genome where the specific oligonucleotidesdefined herein hybridise. In particular, this includes polynucleotideswhich encode the same polypeptide or amino acid sequence but which varyin nucleotide sequence by redundancy of the genetic code. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants.

Nucleic Acid Constructs

The present invention includes nucleic acid constructs comprising thepolynucleotides of the invention, and vectors and host cells containingthese, methods of their production and use, and uses thereof. Thepresent invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides, which when positionedappropriately and connected relative to an expressible genetic sequence,is capable of regulating, at least in part, the expression of thegenetic sequence. Those skilled in the art will be aware that acis-regulatory region may be capable of activating, silencing,enhancing, repressing or otherwise altering the level of expressionand/or cell-type-specificity and/or developmental specificity of a genesequence at the transcriptional or post-transcriptional level. Inpreferred embodiments of the present invention, the cis-acting sequenceis an activator sequence that enhances or stimulates the expression ofan expressible genetic sequence.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide which isapproximately the same as the distance between that promoter and theprotein coding region it controls in its natural setting; i.e., the genefrom which the promoter is derived. As is known in the art, somevariation in this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence element(e.g., an operator, enhancer etc) with respect to a transcribablepolynucleotide to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Promoter” or “promoter sequence” as used herein refers to a region of agene, generally upstream (5′) of the RNA encoding region, which controlsthe initiation and level of transcription in the cell of interest. A“promoter” includes the transcriptional regulatory sequences of aclassical genomic gene, such as a TATA box and CCAAT box sequences, aswell as additional regulatory elements (i.e., upstream activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner. A promoter is usually, butnot necessarily (for example, some PolIII promoters), positionedupstream of a structural gene, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.Promoters may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

“Constitutive promoter” refers to a promoter that directs expression ofan operably linked transcribed sequence in many or all tissues of anorganism such as a plant. The term constitutive as used herein does notnecessarily indicate that a gene is expressed at the same level in allcell types, but that the gene is expressed in a wide range of celltypes, although some variation in level is often detectable. “Selectiveexpression” as used herein refers to expression almost exclusively inspecific organs of, for example, the plant, such as, for example,endosperm, embryo, leaves, fruit, tubers or root. In a preferredembodiment, a promoter is expressed selectively or preferentially inleaves and/or stems of a plant, preferably a cereal plant. Selectiveexpression may therefore be contrasted with constitutive expression,which refers to expression in many or all tissues of a plant under mostor all of the conditions experienced by the plant.

Selective expression may also result in compartmentation of the productsof gene expression in specific plant tissues, organs or developmentalstages. Compartmentation in specific subcellular locations such as theplastid, cytosol, vacuole, or apoplastic space may be achieved by theinclusion in the structure of the gene product of appropriate signals,eg. a signal peptide, for transport to the required cellularcompartment, or in the case of the semi-autonomous organelles (plastidsand mitochondria) by integration of the transgene with appropriateregulatory sequences directly into the organelle genome.

A “tissue-specific promoter” or “organ-specific promoter” is a promoterthat is preferentially expressed in one tissue or organ relative to manyother tissues or organs, preferably most if not all other tissues ororgans in, for example, a plant. Typically, the promoter is expressed ata level 10-fold higher in the specific tissue or organ than in othertissues or organs.

The promoters contemplated by the present invention may be native to thehost plant to be transformed or may be derived from an alternativesource, where the region is functional in the host plant. Other sourcesinclude the Agrobacterium T-DNA genes, such as the promoters of genesfor the biosynthesis of nopaline, octapine, mannopine, or other opinepromoters, tissue specific promoters (see, e.g., U.S. Pat. No. 5,459,252and WO 91/13992); promoters from viruses (including host specificviruses), or partially or wholly synthetic promoters. Numerous promotersthat are functional in mono- and dicotyledonous plants are well known inthe art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkelet al., 1983; Barker et al., 1983); including various promoters isolatedfrom plants and viruses such as the cauliflower mosaic virus promoter(CaMV 35S, 19S). Non-limiting methods for assessing promoter activityare disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989,supra) and U.S. Pat. No. 5,164,316.

Alternatively or additionally, the promoter may be an inducible promoteror a developmentally regulated promoter which is capable of drivingexpression of the introduced polynucleotide at an appropriatedevelopmental stage of the, for example, plant. Other cis-actingsequences which may be employed include transcriptional and/ortranslational enhancers. Enhancer regions are well known to personsskilled in the art, and can include an ATG translational initiationcodon and adjacent sequences. When included, the initiation codon shouldbe in phase with the reading frame of the coding sequence relating tothe foreign or exogenous polynucleotide to ensure translation of theentire sequence if it is to be translated. Translational initiationregions may be provided from the source of the transcriptionalinitiation region, or from a foreign or exogenous polynucleotide. Thesequence can also be derived from the source of the promoter selected todrive transcription, and can be specifically modified so as to increasetranslation of the mRNA.

In an embodiment, the promoter is at least capable of expressing thepolypeptide in leaves of the plant, particularly adult leaves. Examplesof leaf-specific promoters which can be used include those described inYamamoto et al. (1994 and 1997), Kwon et al. (1994), Gotor et al.(1993), Orozco et al. (1993), Matsuoka et al. (1993) and Stockhaus etal. (1987 and 1989).

The nucleic acid construct of the present invention may comprise a 3′non-translated sequence from about 50 to 1,000 nucleotide base pairswhich may include a transcription termination sequence. A 3′non-translated sequence may contain a transcription termination signalwhich may or may not include a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing. Apolyadenylation signal functions for addition of polyadenylic acidtracts to the 3′ end of a mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. Transcriptiontermination sequences which do not include a polyadenylation signalinclude terminators for Poll or PolIII RNA polymerase which comprise arun of four or more thymidines. Examples of suitable 3′ non-translatedsequences are the 3′ transcribed non-translated regions containing apolyadenylation signal from an octopine synthase (ocs) gene or nopalinesynthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983).Suitable 3′ non-translated sequences may also be derived from plantgenes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene, although other 3′ elements known to those of skill in the art canalso be employed.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated 5′ leadersequence (5′UTR), can influence gene expression if it is translated aswell as transcribed, one can also employ a particular leader sequence.Suitable leader sequences include those that comprise sequences selectedto direct optimum expression of the foreign or endogenous DNA sequence.For example, such leader sequences include a preferred consensussequence which can increase or maintain mRNA stability and preventinappropriate initiation of translation as for example described byJoshi (1987).

Vectors

The present invention includes use of vectors for manipulation ortransfer of genetic constructs. By “chimeric vector” is meant a nucleicacid molecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, or plant virus, into which a nucleic acidsequence may be inserted or cloned. A vector preferably isdouble-stranded DNA and contains one or more unique restriction sitesand may be capable of autonomous replication in a defined host cellincluding a target cell or tissue or a progenitor cell or tissuethereof, or capable of integration into the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectormay be an autonomously replicating vector, i.e., a vector that exists asan extrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance gene, aherbicide resistance gene or other gene that can be used for selectionof suitable transformants. Examples of such genes are well known tothose of skill in the art.

The nucleic acid construct of the invention can be introduced into avector, such as a plasmid. Plasmid vectors typically include additionalnucleic acid sequences that provide for easy selection, amplification,and transformation of the expression cassette in prokaryotic andeukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. The actualchoice of a marker is not crucial as long as it is functional (i.e.,selective) in combination with the plant cells of choice. The markergene and the foreign or exogenous polynucleotide of interest do not haveto be linked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al.,1986), which allows for bioluminescence detection, and others known inthe art. By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of, for example, the plant. Accordingly, the nucleic acidcomprises appropriate elements which allow the molecule to beincorporated into the genome, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of aplant cell.

One embodiment of the present invention includes a recombinant vector,which includes at least one polynucleotide molecule of the presentinvention, inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning

Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach,Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin etal., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.Typically, plant expression vectors include, for example, one or morecloned plant genes under the transcriptional control of 5′ and 3′regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The level of a protein, for example Lr34 protein, may be modulated byincreasing the level of expression of a nucleotide sequence that codesfor the protein in a plant cell, or decreasing the level of expressionof a gene encoding the protein in the plant, leading to modifiedpathogen resistance. The level of expression of a gene may be modulatedby altering the copy number per cell, for example by introducing asynthetic genetic construct comprising the coding sequence and atranscriptional control element that is operably connected thereto andthat is functional in the cell. A plurality of transformants may beselected and screened for those with a favourable level and/orspecificity of transgene expression arising from influences ofendogenous sequences in the vicinity of the transgene integration site.A favourable level and pattern of transgene expression is one whichresults in a substantial modification of pathogen resistance or otherphenotype. Alternatively, a population of mutagenized seed or apopulation of plants from a breeding program may be screened forindividual lines with altered pathogen resistance or other phenotypeassociated with pathogen resistance.

Recombinant Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention, or progeny cells thereof.Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Preferred hostcells are plant cells, more preferably cells of a cereal plant, morepreferably barley or wheat cells, and even more preferably a wheat cell.

Transgenic Plants

The term “plant” as used herein as a noun refers to whole plants andrefers to any member of the Kingdom Plantae, but as used as an adjectiverefers to any substance which is present in, obtained from, derivedfrom, or related to a plant, such as for example, plant organs (e.g.leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plantcells and the like. Plantlets and germinated seeds from which roots andshoots have emerged are also included within the meaning of “plant”. Theterm “plant parts” as used herein refers to one or more plant tissues ororgans which are obtained from a plant and which comprises genomic DNAof the plant. Plant parts include vegetative structures (for example,leaves, stems), roots, floral organs/structures, seed (including embryo,cotyledons, and seed coat), plant tissue (for example, vascular tissue,ground tissue, and the like), cells and progeny of the same. The term“plant cell” as used herein refers to a cell obtained from a plant or ina plant and includes protoplasts or other cells derived from plants,gamete-producing cells, and cells which regenerate into whole plants.Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, tubers, pollen, tumor tissue, such as crowngalls, and various forms of aggregations of plant cells in culture, suchas calli. Exemplary plant tissues in or from seeds are cotyledon, embryoand embryo axis. The invention accordingly includes plants and plantparts and products comprising these, particularly seed comprisingmodified oil composition.

As used herein, the term “seed” refers to “mature seed” of a plant,which is either ready for harvesting or has been harvested from theplant, such as is typically harvested commercially in the field, or as“developing seed” which occurs in a plant after fertilization and priorto seed dormancy being established and before harvest.

A “transgenic plant” as used herein refers to a plant that contains anucleic acid construct not found in a wild-type plant of the samespecies, variety or cultivar. That is, transgenic plants (transformedplants) contain genetic material (a transgene) that they did not containprior to the transformation. The transgene may include genetic sequencesobtained from or derived from a plant cell, or another plant cell, or anon-plant source, or a synthetic sequence. Typically, the transgene hasbeen introduced into the plant by human manipulation such as, forexample, by transformation but any method can be used as one of skill inthe art recognizes. The genetic material is preferably stably integratedinto the genome of the plant. The introduced genetic material maycomprise sequences that naturally occur in the same species but in arearranged order or in a different arrangement of elements, for examplean antisense sequence. Plants containing such sequences are includedherein in “transgenic plants”. A “non-transgenic plant” is one which hasnot been genetically modified by the introduction of genetic material byrecombinant DNA techniques. In a preferred embodiment, the transgenicplants are homozygous for each and every gene that has been introduced(transgene) so that their progeny do not segregate for the desiredphenotype.

As used herein, the term “compared to an isogenic plant” refers to aplant which is isogenic relative to the transgenic plant but without thetransgene of interest. Preferably, the corresponding non-transgenicplant is of the same cultivar or variety as the progenitor of thetransgenic plant of interest, or a sibling plant line which lacks theconstruct, often termed a “segregant”, or a plant of the same cultivaror variety transformed with an “empty vector” construct, and may be anon-transgenic plant. “Wild type”, as used herein, refers to a cell,tissue or plant that has not been modified according to the invention.Wild-type cells, tissue or plants may be used as controls to comparelevels of expression of an exogenous nucleic acid or the extent andnature of trait modification with cells, tissue or plants modified asdescribed herein.

Transgenic plants, as defined in the context of the present inventioninclude progeny of the plants which have been genetically modified usingrecombinant techniques, wherein the progeny comprise the transgene ofinterest. Such progeny may be obtained by self-fertilization of theprimary transgenic plant or by crossing such plants with another plantof the same species. This would generally be to modulate the productionof at least one protein defined herein in the desired plant or plantorgan. Transgenic plant parts include all parts and cells of said plantscomprising the transgene such as, for example, cultured tissues, callusand protoplasts.

Plants contemplated for use in the practice of the present inventioninclude both monocotyledons and dicotyledons. Target plants include, butare not limited to, the following: cereals (for example, wheat, barley,rye, oats, rice, maize, sorghum and related crops); beet (sugar beet andfodder beet); pomes, stone fruit and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries andblack-berries); leguminous plants (beans, lentils, peas, soybeans); oilplants (rape or other Brassicas, mustard, poppy, olives, sunflowers,safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts);cucumber plants (marrows, cucumbers, melons); fibre plants (cotton,flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit,mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots,onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon,camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane,tea, vines, hops, turf, bananas and natural rubber plants, as well asornamentals (flowers, shrubs, broad-leaved trees and evergreens, such asconifers). Preferably, the plant is a cereal plant, more preferablywheat, rice, maize, triticale, oats or barley, even more preferablywheat.

As used herein, the term “wheat” refers to any species of the GenusTriticum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. Wheat includes “hexaploid wheat”which has genome organization of AABBDD, comprised of 42 chromosomes,and “tetraploid wheat” which has genome organization of AABB, comprisedof 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T.macha, T. compactum, T. sphaerococcum, T. vavilovii, and interspeciescross thereof. A preferred species of hexaploid wheat is T. aestivum sspaestivum (also termed “breadwheat”). Tetraploid wheat includes T. durum(also referred to herein as durum wheat or Triticum turgidum ssp.durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspeciescross thereof. In addition, the term “wheat” includes potentialprogenitors of hexaploid or tetraploid Triticum sp. such as T. uartu, T.monococcum or T. boeoticum for the A genome, Aegilops speltoides for theB genome, and T. tauschii (also known as Aegilops squamosa or Aegilopstauschii) for the D genome. Particularly preferred progenitors are thoseof the A genome, even more preferably the A genome progenitor is T.monococcum. A wheat cultivar for use in the present invention may belongto, but is not limited to, any of the above-listed species. Alsoencompassed are plants that are produced by conventional techniquesusing Triticum sp. as a parent in a sexual cross with a non-Triticumspecies (such as rye [Secale cereale]), including but not limited toTriticale.

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Hordeum species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Hordeum vulgare or suitablefor commercial production of grain.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide of thepresent invention in the desired plant or plant organ. Transgenic plantscan be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.No. 4,945,050 and U.S. Pat. No. 5,141,131); (3) viral vectors (Clapp,1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediatedmechanisms (Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. A particle delivery system suitable for use with the presentinvention is the helium acceleration PDS-1000/He gun is available fromBio-Rad Laboratories. For the bombardment, immature embryos or derivedtarget cells such as scutella or calli from immature embryos may bearranged on solid culture medium.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135).Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome.

Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations asdescribed (Klee et al., Plant DNA Infectious Agents, Hohn and Schell,(editors), Springer-Verlag, New York, (1985): 179-203). Moreover,technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes and aresuitable for present purposes. In addition, Agrobacterium containingboth armed and disarmed Ti genes can be used for the transformations. Inthose plant varieties where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, Breeding Methods for Cultivar Development, J.Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., Methods for Plant MolecularBiology, Academic Press, San Diego, (1988)). This regeneration andgrowth process typically includes the steps of selection of transformedcells, culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011);Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., 1996); and pea(Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447, WO97/048814, U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541,257, and othermethods are set out in WO 99/14314. Preferably, transgenic wheat orbarley plants are produced by Agrobacterium tumefaciens mediatedtransformation procedures. Vectors carrying the desired nucleic acidconstruct may be introduced into regenerable wheat cells of tissuecultured plants or explants, or suitable plant systems such asprotoplasts. The regenerable wheat cells are preferably from thescutellum of immature embryos, mature embryos, callus derived fromthese, or the meristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis of at least one Lr34 gene or allele that confers enhancedresistance to pathogens to the plant, allows rapid selection of plantscarrying the desired trait, which may be nurtured to maturity in thegreenhouse or field for subsequent further backcrossing to the recurrentparent.

Any molecular biological technique known in the art can be used in themethods of the present invention. Such methods include, but are notlimited to, the use of nucleic acid amplification, nucleic acidsequencing, nucleic acid hybridization with suitably labeled probes,single-strand conformational analysis (SSCA), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavageanalysis (CCM), catalytic nucleic acid cleavage or a combination thereof(see, for example, Lemieux, 2000; Langridge et al., 2001). The inventionalso includes the use of molecular marker techniques to detectpolymorphisms linked to alleles of (for example) Lr34 gene which confersenhanced resistance to plant pathogens. Such methods include thedetection or analysis of restriction fragment length polymorphisms(RFLP), RAPD, amplified fragment length polymorphisms (AFLP) andmicrosatellite (simple sequence repeat, SSR) polymorphisms. The closelylinked markers can be obtained readily by methods well known in the art,such as Bulked Segregant Analysis, as reviewed by Langridge et al.,(2001).

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (M. J. McPherson and S. GMoller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCRcan be performed on cDNA obtained from reverse transcribing mRNAisolated from plant cells expressing a Lr34 gene or allele which confersenhanced resistance to plants pathogens. However, it will generally beeasier if PCR is performed on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,(supra) and Sambrook et al., (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

TILLING

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions I N Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005), and Henikoff etal. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Antibodies

The term “antibody” as used in this invention includes polyclonalantibodies, monoclonal antibodies, bispecific antibodies, diabodies,triabodies, heteroconjugate antibodies, chimeric antibodies includingintact molecules as well as fragments thereof, such as Fab, F(ab′)2, andFv which are capable of binding the epitopic determinant, and otherantibody-like molecules.

The term “specifically binds” refers to the ability of the antibody tobind to at least one polypeptide of the present invention but notsignificantly to known proteins in the sample/organism to be tested.

As used herein, the term “epitope” refers to a region of a polypeptideof the invention which is bound by the antibody. An epitope can beadministered to an animal to generate antibodies against the epitope,however, antibodies of the present invention preferably specificallybind the epitope region in the context of the entire polypeptide.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptideof the invention. Serum from the immunised animal is collected andtreated according to known procedures. If serum containing polyclonalantibodies contains antibodies to other antigens, the polyclonalantibodies can be purified by immunoaffinity chromatography. Techniquesfor producing and processing polyclonal antisera are known in the art.In order that such antibodies may be made, the invention also providespolypeptides of the invention or fragments thereof haptenised to anotherpolypeptide for use as immunogens in animals.

Monoclonal antibodies directed against polypeptides of the invention canalso be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.Panels of monoclonal antibodies produced can be screened for variousproperties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

Other techniques for producing antibodies of the invention are known inthe art.

Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions and the like.

In an embodiment, antibodies of the present invention are detectablylabeled. Exemplary detectable labels that allow for direct measurementof antibody binding include radiolabels, fluorophores, dyes, magneticbeads, chemiluminescers, colloidal particles, and the like. Examples oflabels which permit indirect measurement of binding include enzymeswhere the substrate may provide for a coloured or fluorescent product.Additional exemplary detectable labels include covalently bound enzymescapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. Further, exemplary detectable labels includebiotin, which binds with high affinity to avidin or streptavidin;fluorochromes (e.g., phycobiliproteins, phycoerythrin andallophycocyanins; fluorescein and Texas red), which can be used with afluorescence activated cell sorter; haptens; and the like. Preferably,the detectable label allows for direct measurement in a plateluminometer, for example, biotin. Such labeled antibodies can be used intechniques known in the art to detect polypeptides of the invention.

EXAMPLES Example 1 Materials and Methods Microscopic Analysis ofSeedling Rust Infection

Plants were grown in a growth chamber maintained at 4-8° C. under a 12hour light and dark regime. Seedlings were inoculated at the two leafstage using leaf rust culture 467 and transferred into a humiditychamber (with a temperature range of 16-20° C.) for 24 hours andreturned to the 4-8° C. growth chamber. For microscopic visualisation ofinternal infection structures, the first leaf tissue was autoclaved in1M KOH, washed in 50 mM KPO₄ and stained with a 50 mM KPO₄ (pH 7.8)solution containing 20 ug/ml of wheat germ agglutinin (WGA) conjugatedto the fluorophore alexa 488 (Invitrogen, USA) staining solution. AllWGA-alexa stained tissue was examined under blue light excitation.

RNA Isolation for Semi-Quantitative PCR and Northern Blot

Total RNA was extracted from leaves using a TRIzol solution (38% Phenol,0.8M guanidine thiocyanate, 0.4M ammonium thiocyanate, 0.1M sodiumacetate pH 5 and 10% glycerol). First-strand cDNA for RT-PCR wassynthesized using Superscript II reverse transcriptase (Invitrogen). Aspecific fragment for semi-quantitative RT-PCR of the 5′ end of the PDRwas amplified using the primers Lr34_RT_fl:5′-catcaagatttcaccgcctgtgc-3′ (SEQ ID NO:12) and Lr34_RT_rl:5′-gaagcctagcaacttcacgaggc-3′ (SEQ ID NO:13) at an annealing temperatureof 70° C.

For the Northern blot hybridization analysis, 15 μg of total RNA persample was blotted on a membrane (Hybond-XL, Amersham Biosiences). Theprobe HvS40 (Spielmeyer et al., 2002) was ³²P-labeled at 65° C. usingthe NEBlot® kit (New England BioLabs). Membranes were washed with a0.5×SSC, 0.1% SDS solution at 65° C. and exposed to hypersensitive X-rayfilms (BioMax MS film, Kodak).

Rapid Amplification of cDNA Ends (RACE)

To determine the exact start of the cDNA, a 5′ RACE approach was used.Poly A⁺ RNA was purified from 300 μg of total RNA using the Oligotex®mRNA Mini Kit (Qiagen). Reverse transcription was done using the SMART™RACE cDNA Amplification Kit (Clonetech Laboratories), where an adapterwas ligated to the 5′ end of the cDNA. Amplification of the 5′ end wasdone using an adapter specific primer and the gene specific primerABC_(—)5RACE_r2: 5′-gcggggcccacaatcatctcggc-3′ (SEQ ID NO:14).

Example 2 Genetic Mapping of Lr34 Plant Materials

Three backcross populations were produced and used for genetic mappingof Lr34. The parental parents for the backcrossing, scored phenotypes,population size, and markers mapped on each population are summarized inTable 2.

TABLE 2 The three backcross populations that have been used for thehigh-resolution genetic mapping of Lr34. +Lr34 −Lr34 +Lr34 phenotypicnr. of parent parent backcross line origin scoring plants markers mappedForno Arina Arina Lr34 Swiss winter Leaf tip 1728 BE493812, SWSNP1,(Arina*3/Forno) wheat necrosis, leaf SWSNP2, SWSNP3, rust infectionSWDEL1, SWDEL2, SWDEL3, SWM10, csLVMS PI58548 Thatcher RL6058 ChineseLeaf, stripe, and 1152 Gwm1220, BJ280740, (Thatcher*6/PI58548) landracestem rust; csLVD13, csLVD2, powdery mildew csLVMS, BF473324, csLV34Parula Avocet Avocet Lr34 CIMMYT Leaf and stripe 1152 Gwm1220, csLVD13,(Avocet*5/Parula) rust, leaf tip csLVD2, csLVE17, necrosis csLVMS,csLV34

The ‘Arina×Formo’ fine-mapping population was developed by crossing thehighly resistant Swiss winter wheat cultivar ‘Formo’ with thesusceptible Swiss winter wheat cultivar ‘Arina’. Subsequent backcrossingto Arina and several generations produced by self-fertilization resultedin 103 plants that were “backcross two F4” (BC2F4) containing Lr34 andon average 12.5% of the ‘Formo’ genome in an otherwise Arina geneticbackground. These plants were analyzed for the presence of the Lr34chromosomal segment from ‘Formo’ using two flanking RFLP markersBE493812 and BF473324. One of these plants containing the Lr34 regionwas again crossed to ‘Arina’ and the progeny selfed to produce 1728BC3F2 plants, having on average 6.25% of the ‘Formo’ genome.Recombinants were selected using the two flanking markers BE493812 andSWM10. Phenotyping of the ‘Arina×Formo’ population was done at AgroscopeReckenholz, Zurich, Switzerland during 2006 (BC3F3) and 2007 (BC3F4).Infection rows containing a mixture of susceptible varieties wereinoculated with urediniospores of the Swiss leaf rust isolates Nr.90035, 91047, 93003, 93012, 94015, 95001, 95012, 95028, 95037, 95039,95219, 95251, 96002, 96004, 96209, and 96257. Disease rating was done ontwo replicas.

The Thatcher×RL6058 (Thatcher Lr34) and Avocet×Avocet Lr34 highresolution mapping family, disease evaluation in Australia and atCIMMYT, Mexico were as described in Lagudah et al. (2006) and Spielmeyeret al. (2002). Other genetic stocks used in this work were the nearisogenic lines ‘Thatcher’, ‘Thatcher Lr34’ (=RL6058, Thatcher*6/PI5848),‘Arina’, ‘Arina Lr34’ (Arina*3/Formo).

Marker Development for Genetic Mapping

New molecular markers for mapping were generated by exploiting thesyntenic information of rice, the model grass Brachypodium sylvaticumand the diploid D-genome progenitor Aegilops tauschii as described byBossolini et al. (2006).

To gain physical information of the Lr34 target interval, a partiallyfingerprinted Bacterial Artificial Chromosome (BAC) library of Aegilopstauschii (J. Dvorak, U C Davis) was screened using wheat ESTs related togenes from the syntenic region of rice and Brachypodium sylvaticum.Thirteen BAC clones from three different contigs(HI057C6/HD036L7/HD102K14/HI056G21/HD062G18/HI031F14/HI135B2/RI004I15/RI042I4/HI148C23/BB045B13/HB067N4/BB062G18)were sequenced by low-pass sequencing using an ABI® 3730 sequencer(Applied Biosystems). Sequences were assembled using PHRAP and mined forsimple sequence repeats (SSR). SSRs were amplified by designing primersin the flanking regions (Table 3).

PCR products were analyzed using the LiCOR® DNA Sequencer 4200.Polymorphic SSRs were identified and designated with prefixes “SWM” or“cs”. Sequence tagged sites were developed by designing primers onlow-copy sequences. Locus-specific probes were sequenced and mined forsingle nucleotide polymorphisms (SNP) and insertion/deletions (InDel).Polymorphic SNP-based makers and InDels were designated as Swiss WheatSNP (SWSNP) and Swiss Wheat Deletion (SWDEL), respectively. Primersequences for the PCR based markers mapped on the populations aresummarized in Table 3. Additional low copy probes, csLVD2, csLVD13,csLVE17, for RFLP analysis were isolated from shotgun plasmid librariesfrom the Ae tauschii BAC contigs by screening with total genomic DNAfrom Ae tauschii. Recombinant plasmids where no DNA hybridizationsignals were detected after an overnight exposure were selected aspotential low copy probes.

Using these genetic markers and the mapping populations for Lr34, thehigh-resolution mapping revealed a 0.15 cM target interval for Lr34flanked by genetic markers XSWSNP3 and XcsLVE17 (FIG. 1). Severalmarkers (FIG. 1) were cosegregating with Lr34.

Example 3 Mutagenesis and Isolation of Lr34 Mutants

Seeds of the Lr34 isoline, ‘Lalbahadur Lr34’ were irradiated using a⁶⁰Co source at a dosage of 20 krad and the subsequent M1-M5 generationsevaluated at CIMMYT, Mexico and in Australia as reported in Spielmeyeret al. (2002). Eight mutants were identified from the gamma-irradiatedpopulation. These were analysed using some of the new genetic markers(Example 2). Of the eight mutants, six were interstitial deletionsspanning the Lr34/Yr18/Pm38/Ltn1 locus while the two mutants designatedm19 and m21 showed no loss of markers in the aforementioned geneticlocus. Mutants m19 and m21 were therefore subjected to further analysisutilising the newly identified markers and cosegregating genes.

Sodium azide mutants were developed using seed from a single head of anRL6058 plant grown in the glasshouse to multiply pure seed stocks formutagenesis. Seeds were pre-soaked for 12 hrs at 4° C. before treatingthe grains in an oxygenated solution of 7 mM sodium azide at pH 3.0 for2 hrs. The grains were rinsed and planted in the field. The M2 progenieswere planted as single ear rows and scored for stripe, leaf and stemrust infection in the field in the presence of the pathogens.

TABLE 3 Primer sequences of molecular markers used in this study. markermarker Tm name primer forward primer reverse type [° C.] SWSNP15′-catctttcgtatacatga 5'-gtgtcgattcatgtgag SNP c -> t 60 gaaac-3'(SEQ ID atgc-3' (SEQ ID NO: 15) NO: 16) SWSNP2 5'-cattatgttagcagct5'-ccaaccatcattttggag SNP c -> t 60 tagcg-3' (SEQ ID catg-3' (SEQ IDNO: 17) NO: 18) SWSNP3 5'-gta gat cgt gtc gtg 5'-ctg cta atc cta agtSNP t -> a 65 ttc aac-3' (SEQ ID aac gct c-3' (SEQ ID NO: 19) NO: 20)SWDEL1 5'-cgt gag caa gac atg 5'-gct aca gct ctg aaa   6 bp InDel 66.2ggc g-3' (SEQ ID cta cac-3' (SEQ ID NO: 21) NO: 22) SWDEL25'gat ttg cac gtt gat 5'-cag aat gaa gtt taa   1 bp InDel 60gaa acc ag-3' (SEQ cct ggc ctg-3' (SEQ ID NO: 23) ID NO: 24) SWDEL35'- ggc tgg cta cta cga 5'-atg gtc ttt ttt cct tca 180 bp 65 cga cg-3'(SEQ ID gcc-3' (SEQ ID InDel NO: 25) NO: 26) SWM105'-gcc tac ttt gac ggc 5'-cca tct tga cat act SSR 60 ata tgg-3' (SEQ IDttg gcc ttc c-3' (SEQ (ca)25 NO: 27) ID NO: 28) csLVMS5'-ctc cct ccc gtg agt 5'-atc aaa atc cca ttg SSR 62 ata ttc-3' (SEQ IDcct gac-3' (SEQ ID (at)6tt(at)6 NO: 29) NO: 30) csLV345'-gtt ggt taa gac tgg 5'-tgc ttg cta ttg ctg aat STS 60 tga tgg-3'(SEQ ID agt-3' (SEQ ID NO: 31) NO: 32) SWSNP1_f 5'-cat ctt tcg tat aca5'-gtg tcg att cat gtg SNP c -> t 60 tga gaa ac-3' (SEQ aga tgc-3'(SEQ ID ID NO: 33) NO: 34) SWSNP2_f 5'-cat tat gtt agc agc5'-cca acc atc att ttg SNP c -> t 60 tta gcg-3' (SEQ ID gag cat g-3'(SEQ ID NO: 35) NO: 36) SWSNP3_f 5'-gta gat cgt gtc gtg5'-ctg cta atc cta agt SNP t -> a 65 ttc aac-3' (SEQ ID aac gct c-3'(SEQ ID NO: 37) NO: 38) SWDEL1_f 5'-cgt gag caa gac atg5'-gct aca gct ctg aaa   6 bp InDel 66.2 ggc g-3' (SEQ ID cta cac-3'(SEQ ID NO: 39) NO: 40) SWDEL2_f 5'gat ttg cac gtt gat5'-cag aat gaa gtt taa   1 bp InDel 60 gaa acc ag-3' (SEQ cct ggc ctg-3'(SEQ ID NO: 41) ID NO: 42) SWDEL3_f 5'- ggc tgg cta cta cga5'-atg gtc ttt ttt cct tca 180 bp 65 cga cg-3' (SEQ ID gcc-3' (SEQ IDInDel NO: 43) NO: 44) SWM10f 5'-gcc tac ttt gac ggc5'-cca tct tga cat act SSR (ca)25 60 ata tgg-3' (SEQ ID ttg gcc ttc c-3'(SEQ NO: 45) ID NO: 46)

Six susceptible mutants were isolated and rated 70MS to 90MS for striperust, 50MS to 80MS for leaf rust and 50MS for stem rust under fieldconditions. Two mutants 4C (glycine to glutamic acid at amino acidposition 1030 of SEQ ID NO:1) and 2G (glycine to aspartic acid at aminoacid position 889 of SEQ ID NO:1) were the result of single nucleotidetransitions that resulted in a single amino acid change within thesecond predicted nucleotide binding domain (FIGS. 3 and 5). Thesemutants showed only partial loss of resistance to leaf rust whenexamined microscopically (Example 1). Mutant 2B incorporated a singlenucleotide transition in exon 11 (FIG. 3) that resulted in an early stopcodon. Three mutants 3E, 4E and 2F were the result of single nucleotidetransitions at splice junctions resulting in mis-spliced transcripts.The retention of introns in mutants 3E and 4E introduced early stopcodons near the 5′ end which was predicted to result in a non-functionalprotein. At the microscopic level mutants 3E and 4E were fullysusceptible to leaf rust and indistinguishable from the susceptiblenear-isogenic line ‘Thatcher’. The transcript of mutant 2F lost thesecond last exon (FIG. 3) which was predicted to delete 85 amino acidsfrom the second transmembrane domain. The 2F mutant was more susceptibleto leaf rust than the susceptible control ‘Thatcher’ during the earlyinfection process.

The lack of resistance resulting from loss of a functional Lr34 proteinobserved in the mutation study is consistent with analysis of the Lr34gene from Jagger. Jagger has Lr34-associated alleles of the csLV34marker but is susceptible to leaf rust and stripe rust. Sequencing ofthe Lr34 gene in Jagger identified a G/T point mutation that resulted ina premature stop codon. Consequently, the predicted protein of cultivarJagger lacks 185 amino acids of the C-terminus and this allele is mostlikely not functional. This point mutation probably occurred in aresistant cultivar that carried the +Lr34 allele.

Example 4 Physical Information of the Target Interval and Identificationof the Lr34 Gene

Two BAC libraries of the +Lr34 (resistant) cultivar ‘Chinese Spring’ andthe −Lr34 (susceptible) cultivar ‘Renan’ (INRA, Toulouse, France) werescreened using PCR probes covering the target interval between the twoflanking markers SWSNP3 and csLVE17. The 420 kb physical intervalcontaining both flanking markers was fully sequenced in the resistanthexaploid wheat cultivar ‘Chinese Spring’. To do this, four ‘ChineseSpring’ BAC clones, namely 345C22, 93N17, 1964C18 and 413N16, and the‘Renan’ clone 656106 were selected and fully sequenced at the GenomeSequencing Center, St. Louis, Mo., USA.

Sequence analysis revealed the presence of a gene-rich island containingten open reading frames (FIG. 2) encoding proteins with homologies totwo glycosyl transferases, two cysteine proteinases, two receptor lectinkinases, two cytochrome P450 proteins, a hexose carrier and an ATPbinding cassette (ABC) transporter. None of these genes was present inthe syntenic region in Brachypodium sylvaticum and only the hexosecarrier was found to be conserved in the homologous region on ricechromosome 6 (rice gene Os06g0141000). Significantly and surprisingly,none of the genes appeared to be typical LRR-NBS type genes of the classcommonly associated with pathogen resistance in plants. Therefore, noneof the coding regions was an obvious candidate for encoding Lr34.

To determine whether one of these candidate genes corresponded to Lr34,locus-specific PCR-amplified regions corresponding to the ten candidategenes on each of the eight Lr34 mutants were sequenced. Candidate geneswere amplified by developing locus-specific PCR probes, amplified fromresistant and susceptible cultivars as well as on the eight Lr34mutants, and sequenced. The mutants were the six azide mutants in thegenetic background of ‘Thatcher Lr34’ and two gamma irradiation mutantsin the ‘Lalbahadur Lr34’ background (Example 3).

All of the mutant lines showed a sequence alteration in the open readingframe encoding the ABC transporter (FIG. 3). The three azide mutants 2F,3E and 4E all had a G to A transition at an intron-exon boundary leadingto splice site mutations (FIG. 7, showing retained introns). Transitionsin the two azide induced mutants 2G and 4C resulted in amino acidsubstitutions and line 2B carried a premature stop codon in exon 11. Thetwo gamma irradiation mutants m19 and m21 each showed a 1 bp deletion inexon 10 and 23, respectively, leading to frame shifts and premature stopcodons (FIG. 3).

To remove the possibility of additional mutation sites in the othercosegregating genes, DNA fragments covering 12.7 kb of the other ninecandidate genes and intergenic regions on the four azide mutants 2B, 3E,4C and 4E were sequenced, without finding any additional sequencealterations. Similarly, sequencing showed that the gamma-radiationgenerated mutants m19 and m21 did not harbour any sequence changes inthe coding regions of the remaining nine candidate genes. Therefore, thepossibility that the eight mutations found in the ABC transporter weredue to a very high mutation frequency in these lines could be excluded,and we concluded that the ABC transporter was responsible for conferringthe durable Lr34 disease resistance.

Lr34 co-segregated with partial resistance to adult plant stripe rust(Yr18), powdery mildew (Pm38) as well as leaf tip necrosis (Ltn1). Allof the mutants were more susceptible, as adult plants, to stripe rustand powdery mildew attributed to the loss of Yr18 and Pm38 and alsoexhibited complete or partial loss of Ltn1. These observationsrepresented an important finding, in that eight independent mutationswithin a single ABC transporter gene encoding the Lr34 resistance alsoaccounted for Yr18/Pm38/Ltn1, and demonstrated that a single geneconferred resistance to multiple pathogens.

The protein coding sequence of Lr34 spanned 11.7 kb in the wheat genome.Sequencing of the entire cDNA and comparison of the nucleotide sequencewith the genomic sequence (SEQ ID NO:3) revealed that Lr34 had 24 exons.The gene contained 23 introns including a large intron of 2.5 kb betweenexons 4 and 5 (FIG. 3). The protein encoded by Lr34 from the resistantcultivar Chinese Spring had 1401 amino acids (SEQ ID NO:1), while theprotein from the susceptible cultivar Renan had 1402 amino acids (SEQ IDNO:4, FIG. 4). Comparison of the amino acid sequence with other ABCtransporters showed that the Lr34 proteins belonged to the PleiotropicDrug Resistance (PDR) subfamily of ABC transporters. PDRs share a commonbasic structure containing two distinct domains: a cytosolic nucleotidebinding domain (NBD) that contains the conserved motifs “Walker A” and“Walker B” involved in ATP binding and hydrolysis, and a hydrophobictransmembrane domain (TMD) involved in translocating the substrate. Bothdomains are present in duplicate, therefore the structure of PDRs isdesignated [NBD-TMD]₂ (FIG. 5).

The PDR family is only found in fungi and plants. Fifteen PDR-like geneshave been identified in the genome of Arabidopsis and 23 members weredescribed in rice (Crouzet et al., 2006). It is known that PDRs conferresistance to various drugs, but little is known about the substratespecificity of this protein class (Rogers et al., 2001). It haspreviously been reported that PEN3/PDR8, a PDR from Arabidopsis,contributes to nonhost resistance to pathogens (Stein et al., 2006). Theclosest Lr34 homolog in rice is PDR23, showing 88% identity on the aminoacid level (Table 4). In Arabidopsis, Lr34 shows closest homology to thetwo transporters PDR5 and PDR9, with 56% identity. The alignment ofthese amino acid sequences is shown in FIG. 6.

TABLE 4 Percentage amino acid identity of wheat Lr34 to homologs of Lr34from other plant species. SPECIES GenBank Accession No. % Identity RiceEAZ20654 78 EAY83289 76 CAD59575 55 Tobacco CAH39853 (NtPDR3) 56 GrapeCAN65735 56 Arabidopsis NP_181265 (PDR5) 56 NP_190916 (PDR9) 55 DAA00881(PDR13) 54 DAA00869 (PDR2) 52 NP_176196 (PDR8/PEN3) 50

The present inventors next determined the sequence differences betweenthe Lr34 alleles in cultivars with or without Lr34-based resistance.Comparison of genomic sequences of the PDR in the +Lr34 cultivar‘Chinese Spring’ and the −Lr34 French winter wheat cultivar ‘Renan’revealed that the gene was present in both wheat varieties. There wereonly three polymorphisms in the coding sequences between these two lines(FIG. 3). One SNP was located in the large intron 4. The other twosequence alterations were located in exons 11 and 12. A deletion ofthree base pairs ‘TTC’ found in exon 11 in ‘Chinese Spring’ results inthe deletion of a phenylalanine residue whereas a second SNP in exon 12converted a tyrosine to a histidine in the resistant cultivar. Bothsequence differences located in exons affect the first transmembranedomain connecting the two nucleotide binding domains and they may alterthe structure and binding specificity of the transporter (FIG. 4).Sequence comparison of 2 kb of the putative promoter regions did notreveal any differences between the resistant and susceptible alleles.

To find out which of these three sequence differences were required fordetermining the resistance, their diagnostic value was determined on aset of +/−Lr34 genotypes sourced from different Lr34 breeding lineages(Table 5). All the +Lr34 lines showed the same haplotype as ‘ChineseSpring’ and all the −Lr34 lines were identical to that of ‘Renan’.Hence, all of the three reported sequence differences may be importantfor determining the resistance conferred by Lr34, although we have noevidence that the SNP in intron 4 affects the splice efficiency ofeither of the alleles. Given that the same haplotype was found in theLr34 PDR-ABC transporter gene for the spring wheats from the South/NorthAmerican breeding programs, winter wheats from Europe and the orientalLr34 genotypes (Table 5), we infer that a single event likely accountsfor the origin of Lr34 in a wheat landrace. Evidence linking theAmerican and European wheats containing Lr34 is traced back to thefounder sib cultivars, ‘Mentana’ and ‘Ardito’ developed at the beginningof the last century (Kolmer et al., 2008).

When testing the diagnostic potential of the SNP located in intron 4 athird allele was identified. The winter wheat cultivars Zinal, Allalinand Galaxie, as well as the spelt (Triticum spelta) varieties Ostro andRouquin showed the +Lr34 haplotype in intron 4, but had the −Lr34haplotype for the two markers in exons 11 and 12. Hence, these linesform a third haplotype. Interestingly, the reciprocal allele (T, for SNPin intron 4 and +Lr34 for both exon markers) was never observed. Thisfinding suggests that this haplotype arose through mutation rather thanrecombination and probably represents the progenitor of the functional+Lr34 haplotype.

TABLE 5 Polymorphisms in Lr34 alleles of wheat genotypes. +/− A/T C/TGenotype Origin Lr34 SNP TTC/DEL SNP Chinese Spring China + A DEL CRL6058* China + A DEL C Fukuho Japan + A DEL C Mentana Italy + DEL CFrontana Brazil + A DEL C Frontierra Brazil − T TTC T Ardito Italy + ADEL C JupatecoR CIMMYT + A DEL C JupatecoS CIMMYT − T TTC T GlenleaCanada + A DEL C Thatcher Canada − T TTC T Anza USA + A DEL C ChrisUSA + A DEL C Condor Australia + A DEL C Penjamo 62 CIMMYT + Inia66CIMMYT − LalbahadurLr34 CIMMYT + A DEL C Lalbahadur India − T TTC TForno Switzerland + Arina Switzerland − Pegaso Italy + A DEL C BezostajaRussia + A DEL C Kavkaz Russia + A DEL C Roazon France − Capelle DesprezUK − T TTC T Maris Huntsman UK − T TTC T Renan France − T TTC T“Synthetic”_taus − T TTC T AL8/78_taus Armenia − T TTC T AUS18913_tausIran − T TTC T

Example 5 Expression of Lr34

Lr34 is a model for adult plant resistance, which is not effective atthe seedling stage under normal field conditions. To determine whetherthis was related to lower expression of Lr34 at the seedling stage,semi-quantitative RT-PCR was used to measure expression levels atvarious stages of plant development using the near isogenic lines‘Thatcher’ and ‘Thatcher Lr34’. The PDR was expressed at very low levelsin 14 days old seedlings grown at 20° C. whereas the expression levelwas significantly higher in flag leaves of adult plants after 53 and 63days (FIG. 7). There was no substantial difference in expression betweenresistant and susceptible plants which was in agreement with the findingthat there were no sequence differences in the promoter regions of theresistant and susceptible alleles. Interestingly, the unspliced productwas observed to accumulate in adult plants after 63 days. Also, analtered transcript in ‘ThatcherLr34’ had 92 nucleotides missing fromexon 10 which was predicted to disrupt the reading frame and result in atruncated protein.

It has been shown that Lr34 confers resistance at the seedling stage toleaf rust cultures at low temperatures (Dyck and Samborski, 1982).Analysis of the mutants and the parental Lr34 lines grown, as seedlings,at low temperatures (4-8° C.) and infected with leaf rust revealed a“slow rusting” resistance response with the intact Lr34 gene. In theinitial 2-3 weeks post infection, differences in colonized mesophylcells between mutants m19, m21 and lalbahadur Lr34′ were insignificant.However by the fifth week the colonized area had extended at least fourtimes in size with mutants m19 and m21 when compared with the activeLr34 gene. External symptoms of sporulation in seedlings were evident inthe mutants by the fifth week whereas the presence of the active Lr34gene delayed visible symptoms until after the sixth week post infection.This observation was akin to the longer latency period that wascharacteristic of the slow rusting mechanism of Lr34 resistance.

Lr34 conferred a broad spectrum resistance against several obligatebiotrophic pathogens including fungi from the Ascomycetes andBasidiomycetes. Rubiales and Nicks (1995) reported that Lr34 wasassociated with reduced intercellular hyphal growth but not with ahypersensitive response or papilla formation. The eight Lr34 mutantswere affected in their resistance against leaf rust, stripe rust andpowdery mildew and they did not show leaf tip necrosis as describedabove. Infection experiments revealed that the level of resistance wascoupled to the development of leaf tip necrosis and that artificialinoculation with leaf rust before emergence of leaf tip necrosis led tomore severe disease symptoms than infections at later time points. Theseobservations suggested that the resistance mechanism of Lr34 was due toa general physiological effect rather than to a ‘classical’ resistancemechanisms involving recognition of pathogen elicitors or secretion ofantifungal components.

From this, a hypothesis was formed that the durable resistance conferredby Lr34 was associated with and at least partly due to prematuresenescence of the flag leaf, in particular the leaf tips. In contrast tonecrosis, senescence is a highly controlled process including theremobilization of nutrients and the degradation of chlorophyll. It wasconsidered that premature leaf senescence starting from the leaf tipcould hamper the feeding of the pathogen from host cells and mightretard its growth and multiplication. Senescence-related genes weretherefore analysed in the wheat plants with or without Lr34.

The gene HvS40 was known to be highly upregulated during senescence inbarley (Krupinska et al., 2007). A probe corresponding to this gene wasprepared from cDNA. Using this probe in a Northern blot hybridizationanalysis revealed that wheat HvS40 was highly expressed in flag leaftips of ‘Thatcher Lr34’ but not of ‘Thatcher’ in 63 days old plants.Furthermore the gene was down-regulated or not expressed in the six Lr34azide mutants (FIG. 8). This was strong evidence that Lr34 regulatedsenescence of flag leaves in adult wheat plants. On the other hand,microscopic observations have indicated the build up of cell wallappositions following leaf rust infection of Lr34 genotypes. It istherefore likely that Lr34 mediated resistance affected pathogendevelopment in a more complex way.

The cloning of Lr34 is the first reported cloning of a multi-pathogenresistance QTL from wheat, which includes Lr34, Yr18, Pm38, Ltn1 anddemonstrated this was controlled by a single gene. An ABC transporter ofthe PDR subfamily was identified as the gene being responsible forconferring this durable adult plant resistance. Resistant andsusceptible alleles differed by only three minor sequence alterationswithin the coding sequence. The resistant allele was thought toaccelerate senescence of flag leaf tips and therefore compromisenutrient uptake by obligate biotrophic pathogens.

Example 6 Related Genes from Wheat and Other Species

The homoeologous genes from the A and B genomes of wheat, and genesencoding homologs in other species were isolated by using probes derivedfrom the wheat Lr34 gene to probe cDNA or genomic libraries. Thehomoeologous genes from the A and B genomes were isolated. A homologousgene was isolated from Aegilops tauschii, a diploid cereal (D genome)related to wheat (SEQ ID NO:6). Other related sequences were identifiedfrom EST databases, containing partial sequences (Table 6).

TABLE 6 ESTs which are homologous to Lr34. The percentage sequenceidentity over the matching region is shown. Region of SEQ ID NO: 2 EST'sIdentity corresponding to the EST Wheat CJ669561 99% 1496-2333 DR73373496% 3089-3802 CJ562397 99% 3561-4206 CV773074 100%  3732-4206 RiceAK102367 91%  569-2775 AK103110 91%  569-2775 CB630740 91% 1280-2085CI097424 92% 2292-2775 CI380443 93% 2425-2775 CI361087 93% 2432-2775CI522302 90% 1904-2252 Barley BU991506 71% 2518-2991 Sugarcane CA07585977% 3216-3883 CA267101 77% 3407-3995

A related gene member was also detected in barley when a cDNA probederived from the 3′ half of the gene was hybridised to genomic barleyDNA under standard conditions.

The present inventors have also determined the homeolog of Lr34 presenton chromosome 7B of wheat. The protein sequence of this homeolog isprovided as SEQ ID NO:63 and the cDNA sequence as SEQ ID NO:64.

Example 7 Production of Transgenic Wheat Expressing an Exogenous AdultPlant Pathogen Resistance Gene

In order to produce transgenic wheat, the polynucleotide comprising asequence of nucleotides as provided in SEQ ID NO:2 is sub-cloned into apPlex vector (Schunmann et al., 2003) such that the subterranean cloverstunt virus promoter is able to drive gene transcription in a wheatcell.

Transformation of wheat embryos from the cultivar Bobwhite 26 isperformed according to the method of Pellegrineschi et al. (2002). Toconfirm that the plants that were produced contained the construct, PCRanalysis is performed on genomic DNA extracted from leaves using aFastDNA® kit (BIO 101 Inc., Vista, Calif., USA) according to thesuppliers instructions. The DNA is eluted into 100 μl sterile deionizedwater and 1μl used in PCR.

Plants are tested for enhanced resistance to plant pathogens such asPuccinia graminis f. sp. tritici (which causes stem rust), Pucciniastriiformis (which causes stripe rust) and/or Puccinia recondite f. sp.tritici (which causes leaf rust).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from AU 2008904364 filed 25 Aug.2008, the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1.-39. (canceled)
 40. A transgenic cereal plant which has integratedinto its genome an exogenous polynucleotide encoding an Lr34 adult plantpathogen resistance polypeptide comprising an amino acid sequence atleast 95% identical to the amino acid sequence set forth as SEQ ID NO:1,wherein the adult plant pathogen resistance polypeptide providesenhanced resistance to a plant pathogen when compared to an isogeniccereal plant lacking the exogenous polynucleotide, and wherein thepathogen is one or more of Bipolaris sorokiniana, Erysiphe graminis f.sp. tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis andPuccinia recondita f. sp. tritici.
 41. The transgenic cereal plant ofclaim 40 which is a wheat plant.
 42. The transgenic cereal plant ofclaim 40, wherein the amino acid sequence of the Lr34 adult plantpathogen resistance polypeptide is at least 98% identical to the aminoacid sequence set forth as SEQ ID NO:1.
 43. The transgenic cereal plantof claim 40, wherein the amino acid sequence of the Lr34 adult plantpathogen resistance polypeptide comprises the amino acid sequence setforth as SEQ ID NO:1.
 44. The transgenic cereal plant of claim 43 whichis a wheat plant.
 45. A transgenic cereal seed which has integrated intoits genome an exogenous polynucleotide encoding an Lr34 adult plantpathogen resistance polypeptide comprising an amino acid sequence atleast 95% identical to the amino acid sequence set forth as SEQ ID NO:1,wherein the adult plant pathogen resistance polypeptide providesenhanced resistance to a cereal plant produced from the seed to a plantpathogen when compared to an isogenic cereal plant lacking the exogenouspolynucleotide, and wherein the pathogen is one or more of Bipolarissorokiniana, Erysiphe graminis f. sp. tritici, Puccinia graminis f. sp.tritici, Puccinia striiformis and Puccinia recondita f. sp. tritici. 46.The transgenic cereal seed of claim 45 which is a wheat seed.
 47. Thetransgenic cereal seed of claim 45, wherein the amino acid sequence ofthe Lr34 adult plant pathogen resistance polypeptide is at least 98%identical to the amino acid sequence set forth as SEQ ID NO:1.
 48. Thetransgenic cereal seed of claim 45, wherein the amino acid sequence ofthe Lr34 adult plant pathogen resistance polypeptide comprises the aminoacid sequence set forth as SEQ ID NO:1.
 49. The transgenic cereal seedof claim 48 which is a wheat seed.
 50. A method of producing atransgenic cereal plant which has integrated into its genome anexogenous polynucleotide encoding an Lr34 adult plant pathogenresistance polypeptide comprising an amino acid sequence at least 95%identical to the amino acid sequence set forth as SEQ ID NO:1, themethod comprising i) introducing a polynucleotide encoding the Lr34adult plant pathogen resistance polypeptide into a cereal plant cell,wherein the polynucleotide is operably linked to a promoter, and ii)generating a transgenic cereal plant from the cell, thereby producingthe transgenic cereal plant, wherein the adult plant pathogen resistancepolypeptide provides enhanced resistance to the plant to a plantpathogen when compared to an isogenic cereal plant lacking the exogenouspolynucleotide, and wherein the pathogen is one or more of Bipolarissorokiniana, Erysiphe graminis f. sp. tritici, Puccinia graminis f. sp.tritici, Puccinia striiformis and Puccinia recondite f. sp. tritici. 51.The method of claim 50, wherein the cereal plant is a wheat plant. 52.The method of claim 50, wherein the amino acid sequence of the Lr34adult plant pathogen resistance polypeptide is at least 98% identical tothe amino acid sequence set forth as SEQ ID NO:1.
 53. The method ofclaim 50, wherein the amino acid sequence of the Lr34 adult plantpathogen resistance polypeptide comprises the amino acid sequence setforth as SEQ ID NO:1.
 54. The method of claim 53, wherein the cerealplant is a wheat plant.
 55. A method of producing a transgenic cerealplant part which has integrated into its genome an exogenouspolynucleotide encoding an Lr34 adult plant pathogen resistancepolypeptide comprising an amino acid sequence at least 95% identical tothe amino acid sequence set forth as SEQ ID NO:1, the method comprising,a) growing the transgenic cereal plant of claim 1, and b) harvesting atransgenic cereal plant part from the plant.
 56. The method of claim 55,wherein the cereal plant is a wheat plant.
 57. The method of claim 55,wherein the amino acid sequence of the Lr34 adult plant pathogenresistance polypeptide is at least 98% identical to the amino acidsequence set forth as SEQ ID NO:1.
 58. The method of claim 55, whereinthe amino acid sequence of the Lr34 adult plant pathogen resistancepolypeptide comprises the amino acid sequence set forth as SEQ ID NO:1.59. The method of claim 58, wherein the cereal plant is a wheat plant.